Vitamin B3, and other B-vitamins such as thiamine (vitamin B1), riboflavin (vitamin B2), and pyridoxine (vitamin B6) are extracted in their coenzyme forms from foodstuffs. During digestion, the coenzymes are catabolized to the free circulating vitamins, which are then passively or actively transported across membranes, and salvaged intracellularly to their respective cofactors, Mammals are entirely reliant on a dietary source of vitamin B1 and heavily dependent on the dietary supply of vitamins B2, B3, and B6. Of note, acute deficiencies in vitamin B1 and vitamin B3 affect identical organs, with identical outcomes if left untreated: dementia and death. Conditions such as diabetes and obesity, alcoholism, a high fat diet, and conditions where therapy impacts nutrition can compromise suitable absorption of these vitamins.
The dietary vitamin B3, which encompasses nicotinamide (“Nam” or “NM”), nicotinic acid (“NA”), and nicotinamide riboside (“NR”), is a precursor to the coenzyme nicotinamide adenine dinucleotide (“NAD+”), its phosphorylated parent (“NADP+” or “NAD(P)+”), and their respective reduced forms (“NADH” and “NADPH,” respectively).
Eukaryotes can synthesize NAD+ de novo via the kynurenine pathway from tryptophan. See W. A. Krehl et al., Growth-retarding Effect of Corn in Nicotinic Acid-Low Rations and its Counteraction by Tryptophane, 101 SCIENCE 489 (1945); Gunther Schutz & Philip Feigelson, Purification and Properties of Rat Liver Tryptophan Oxygenase, 247 J. BIOL. CHEM. 5327 (1972); each of which is incorporated by reference herein in its entirety. The kynurenine pathway is a de novo pathway that originates from tryptophan. Through the sequential enzymatic action of tryptophan 2,3-dioxygenase (“TDO”), indoleamine 2,3-dioxygenase (“IDO”), kynurenine formamidase (“KFase”), kynurenine 3-hydroxylase (“K3H”), kynureninase, and 3-hydroxyanthranylate 3,4-dioxygenase (“3HAO”), tryptophan (“Trp”) is converted to quinolinic acid (“QA”). See Javed A. Khan et al., Nicotinamide adenine dinucleotide metabolism as an attractive target for drug discovery, 11 EXPERT OPIN. THER. TARGETS 695 (2007), incorporated by reference herein in its entirety. Quinolinic acid (QA) is converted to nicotinic acid mononucleotide (“NaMN”) through the action of quinolinic phosphoribosyltransferase (“QAPRTase”). See Khan et al., 2007.
The de novo kynureninase pathway, which produces nicotinic acid mononucleotide (NaMN) from quinolinic acid (QA), feeds into the well-established Preiss-Handler pathway, in which nicotinic acid mononucleotide (NaMN) is an intermediate. The Preiss-Handler pathway is a salvage pathway that starts with the conversion of nicotinic acid (NA) to nicotinic acid mononucleotide (NaMN), catalyzed by the enzyme nicotinate phosphoribosyltransferase (“NAPRT” or “NAPRTase”). Nicotinic acid mononucleotide (NaMN) is then adenylylated to form nicotinic acid adenine dinucleotide (“NaAD”), catalyzed by the enzyme nicotinic acid/nicotinamide mononucleotide adenylyltransferase (“NMNAT”). Nicotinic acid adenine dinucleotide (NaAD) is in turn amidated to form nicotinamide adenine dinucleotide (NAD+), catalyzed by the enzyme nicotinamide adenine dinucleotide synthetase (“NADS”). Nicotinamide (Nam or NM), which is a breakdown product of NAD+, can be converted to nicotinic acid (NA), catalyzed by the enzyme nicotinamide deamidase (“NM deamidase”). See Jack Preiss & Philip Handler, Biosynthesis of Diphosphopyridine Nucleotide, 233 J. BIOL. CHEM. 493 (1958), incorporated by reference herein in its entirety. See also Khan et al., 2007.
Another salvage pathway can convert nicotinamide (Nam or NM), the breakdown product of nicotinamide adenine dinucleotide (NAD+), into nicotinamide mononucleotide (“NMN”), by the action of the enzyme nicotinamide phosphoribosyltransferase (“NMPRT” or “NMPRTase”). Nicotinamide mononucleotide (NMN) can then be directly converted into nicotinamide adenine dinucleotide (NAD+) by nicotinic acid/nicotinamide mononucleotide adenylyltransferase (NMNAT). Alternatively, nicotinamide (Nam or NM) can be deamidated to form nicotinic acid (NA), which can then enter the Preiss-Handler pathway. Analysis of genome sequences suggests that the above two salvage pathways are often mutually exclusive; many organisms contain either NM deamidase or NMPRTase. See Khan et al., 2007.
Nicotinamide riboside (NR) can also be used as a precursor for nicotinamide adenine dinucleotide (NAD+) biosynthesis, and nicotinamide riboside kinase (“NRK”) catalyzes the phosphorylation of nicotinamide riboside (NR) to produce nicotinamide mononucleotide (NMN). See Khan et al., 2007.
Notably, nicotinamide riboside (NR) has not been considered a precursor to nicotinamide adenine dinucleotide (NAD+) via the Preiss-Handler salvage pathway, or via conversion into nicotinic acid mononucleotide (NaMN) or nicotinic acid adenine dinucleotide (NaAD) as intermediates. Instead, the biosynthetic pathway for nicotinic acid riboside (NAR) is known to proceed directly to nicotinic acid mononucleotide (NaMN), then nicotinic acid adenine dinucleotide (NaAD), and ultimately to form NAD+.
Nicotinamide adenine dinucleotide (NAD+) is an enzyme co-factor and the central reduction-oxidation coenzyme that is essential for the function of several enzymes related to reduction-oxidation reactions and cellular energy metabolism, See Peter Belenky et al., NAD+ metabolism in health and disease, 32 TRENDS IN BIOCHEMICAL SCIS. 12 (2007); Katrina L. Bogan & Charles Brenner, Nicotinic Acid, Nicotinamide, and Nicotinamide Riboside: A Molecular Evaluation of NAD+Precursor Vitamins in Human Nutrition, 28 ANNUAL REV. OF NUTRITION 115 (2008); each of which is incorporated by reference herein in its entirety. Nicotinamide adenine dinucleotide (NAD+) functions as an electron carrier or hydride group acceptor in cell metabolism, forming reduced nicotinamide adenine dinucleotide (NADH), with concomitant oxidation of metabolites derived from carbohydrates, amino acids, and fats. See Bogan & Brenner, 2008. The NAD+/NADH ratio controls the degree to which such reactions proceed in oxidative versus reductive directions. Whereas fuel oxidation reactions require NAD+ as a hydride acceptor, the processes of gluconeogenesis, oxidative phosphorylation, ketogenesis, detoxification of reactive oxygen species, and lipogenesis require reduced co-factors, NADH and NADPH, to act as hydride donors.
In addition to its role as a coenzyme, NAD+ is the consumed substrate, and thus activator, of enzymes such as: poly-ADP-ribose polymerases (“PARPs”); sirtuins, a family of protein deacetylases that have been implicated in metabolic function and extended lifespan in lower organisms; and cyclic ADP-ribose synthetases. See Laurent Mouchiroud et al., The NAD+/Sirtuin Pathway Modulates Longevity through Activation of Mitochondrial UPR and FOXO Signaling, 154 CELL 430 (2013), incorporated by reference herein in its entirety. See also Belenky et al., 2006. The co-enzymatic activity of NAD+, together with the tight regulation of its biosynthesis and bioavailability, makes it an important metabolic monitoring system that is clearly involved in the aging process.
Once converted intracellularly to NADP+, vitamin B3 is used as a co-substrate in two types of intracellular modifications, which control numerous essential signaling events (adenosine diphosphate ribosylation and deacetylation), and is a cofactor for over 400 reduction-oxidation enzymes, thus controlling metabolism. This is demonstrated by a range of metabolic endpoints including the deacetylation of key regulatory proteins, increased mitochondrial activity, and oxygen consumption. Critically, the NADPH-cofactor family can promote mitochondrial dysfunction and cellular impairment if present in sub-optimal intracellular concentrations. Vitamin B3 deficiency yields to evidenced compromised cellular activity through NAD+ depletion.
In reduction-oxidation reactions, the nucleotide structures of NAD+, NADH, NADP+, and NADPH are preserved. In contrast, PARP, sirtuin, and cyclic ADP-ribose synthetase activities hydrolyze the glycosidic linkage between the nicotinamide (Nam or NM) and the ADP-ribosyl moieties of NAD+ to signal DNA damage, alter gene expression, control post-translational modifications, and regulate calcium signaling.
In animals, NAD+-consuming activities and cell division necessitate ongoing NAD+ synthesis, either through the de novo pathway that originates with tryptophan, or via the salvage pathways from NAD+-precursor vitamins nicotinamide (Nam or NM), nicotinic acid (NA), and nicotinamide riboside (NR). See Bogan & Brenner, 2008. Dietary NAD+ precursors, which include tryptophan and the three NAD+-precursor vitamins, prevent pellagra, a disease characterized by dermatitis, diarrhea, and dementia. The beneficial effect of additional NAD+ bioavailability through nicotinamide (Nam or NM), nicotinic acid (NA), and nicotinamide riboside (NR) supplementation is primarily observed in cells and tissues where metabolism and mitochondrial function had been compromised.
Interestingly, supplementation with nicotinic acid (NA) and nicotinamide (Nam or NM), while critical in acute vitamin B3 deficiency, does not demonstrate the same physiological outcomes compared with that of nicotinamide riboside (NR) supplementation, even though, at the cellular level, all three metabolites are responsible for NAD+ biosynthesis. This emphasizes the complexity of the pharmacokinetics and bio-distribution of B3-vitamin components. The bulk of intracellular NAD+ is believed to be regenerated via the effective salvage of nicotinamide (Nam or NM), while de novo NAD+ is obtained from tryptophan. See Anthony Rongvaux et al., Reconstructing eukaryotic NAD metabolism, 25 BIOESSAYS 683 (2003), incorporated by reference herein in its entirety. Crucially, these salvage and de novo pathways depend on the functional forms of vitamin B1, B2, and B6 to generate NAD+ via a phosphoriboside pyrophosphate intermediate. Nicotinamide riboside (NR) is the only form of vitamin B3 from which NAD+ can be generated in a manner independent of vitamin B1, B2, and B6, and the salvage pathway using NR for the production of NAD+ is expressed in most eukaryotes.
Thiamine (vitamin B1), riboflavin (vitamin B2), niacin (vitamin B3), and pyridoxine (vitamin B6) are salvaged from food and converted back intracellularly to their respective, bioactive forms: Thiamine DiPhosphate (“ThDP”); Flavin Adenine Dinucleotide (“FAD”); Nicotinamide Adenine Dinucleotide (NAD+); and PyridoxaL Phosphate (“PLP”). The conversion of vitamins B1, B2, and B6 to ThDP, FAD, and PLP, respectively, is ATP-dependent. Two of the three salvage pathways that convert vitamin B3 to NAD+ are dependent on ThDP (B1), with the de novo production of NAD+ from tryptophan depending on the bioactive forms of vitamins B1, B2, and B6. The vitamin B1 dependency comes from the fact that ThDP (B1) is cofactor for the transketolases involved in the biosynthesis of phosphoriboside pyrophosphate, an essential substrate in these aforementioned NAD+ salvage and de novo pathways. The most recently identified, yet so far believed redundant, third NAD+ salvage pathway, the Nicotinamide Riboside (NR) dependent NAD+ biosynthetic pathway, does not require phosphoriboside pyrophosphate and is independent of vitamins B1, B2, and B6.
Though nicotinamide riboside (NR) is present in milk, the cellular concentrations of NAD+, NADH, NADP+, and NADPH are much higher than those of any other NAD+ metabolites, such that dietary NAD+ precursor vitamins are largely derived from enzymatic breakdown of NAD+. See Pawel Bieganowski & Charles Brenner, Discoveries of Nicotinamide Riboside as a Nutrient and Conserved NRK Genes Establish a Preiss-Handler Independent Route to NAD+ in Fungi and Humans, 117 CELL 495 (2002); Charles Evans et al., NAD+metabolite levels as a function of vitamins and calorie restriction: evidence for different mechanisms of longetivity, 10 BMC CHEM. BIOL. 2 (2010); Samuel A. J. Trammell & Charles Branner, Targeted, LCMS-Based Metabolomics for Quantitative Measurement of NAD+Metabolites, 4 COMPUTATIONAL & STRUCTURAL BIOTECH. J. 1 (2013); each of which is incorporated by reference herein in its entirety. Put another way, though milk is a source of nicotinamide riboside (NR), the more abundant sources of nicotinamide riboside (NR), nicotinamide (Nam or NM), and nicotinic acid (NA) are any whole foodstuffs in which cellular NAD+ is broken down to these compounds. Human digestion and the microbiome play roles in the provision of these vitamins in ways that are not fully characterized.
Different tissues maintain NAD+ levels through reliance of different biosynthetic routes. See Federica Zamporlini et al., Novel assay for simultaneous measurement of pyridine mononucleotides synthesizing activities allows dissection of the NAD+ biosynthetic machinery in mammalian cells, 281 FEBS J. 5104 (2014); Valerio Mori et al., Metabolic Profiling of Alternative NAD Biosynthetic Routes in Mouse Tissues, 9 PLOS ONE e113939 (2014); each of which is incorporated by reference herein in its entirety. Because NAD+-consuming activities frequently occur as a function of cellular stresses and produce nicotinamide (Nam or NM), the ability of a cell to salvage nicotinamide (Nam or NM) into productive NAD+ synthesis through nicotinamide phosphoribosyltransferase (“NAMPT”) activity versus methylation of nicotinamide (Nam or NM) to N-methylnicotinamide (“MeNam”) regulates the efficiency of NAD+-dependent processes. See Charles Brenner, Metabolism: Targeting a fat-accumulation gene, 508 NATURE 194 (2014); Véronique J. Bouchard et al., PARP-1, a determinant of cell survival in response to DNA damage, 31 EXPERIMENTAL HEMATOLOGY 446 (2003); each of which is incorporated by reference herein in its entirety. NAD+ biosynthetic genes are also under circadian control, and both NAMPT expression and NAD+ levels are reported to decline in a number of tissues as a function of aging and overnutrition. See Kathryn Moynihan Ramsey et al., Circadian Clock Feedback Cycle Through NAMPT-Mediated NAD+ Biosynthesis, 324 SCIENCE 651 (2009); Yasukazu Nakahata et al., Circadian Control of the NAD+ Salvage Pathway by CLOCK-SIRT1, 324 SCIENCE 654 (2009); Jun Yoshino et al., Nicotinamide Mononucleotide, a Key NAD+ Intermediate Treats the Pathophysiology of Diet- and Age-Induced Diabetes in Mice, 14 CELL METABOLISM 528 (2011); Ana P. Gomes et al., Declining NAD+ Induces a Pseudohypoxic State Disrupting Nuclear-Mitochondrial Communication during Aging, 155 CELL 1624 (2013); Nady Braidy et al., Mapping NAD+ metabolism in the brain of ageing Wistar rats: potential targets for influencing brain senescence, 15 BIOGERONTOLOGY 177 (2014); Eric Verdin, NAD+ in aging, metabolism, and neurodegeneration, 350 SCIENCE 1208 (2015); each of which is incorporated by reference herein in its entirety.
High-dose nicotinic acid (NA), but not high-dose nicotinamide (Nam or NM), has been used by people for decades to treat and prevent dyslipidemias, though its use is limited by painful flushing. See Joseph R. DiPalma & William S. Thayer, Use of Niacin as a Drug, 11 ANNUAL REV. OF NUTRITION 169 (1991); Jeffrey T. Kuvin et al., Effects of Extended-Release Niacin on Lipoprotein Particle Size, Distribution, and Inflammatory Markers in Patients With Coronary Artery Disease, 98 AM. J. OF CARDIOLOGY 743 (2006); each of which is incorporated by reference herein in its entirety. Though only approximately 15 milligrams per day of either nicotinic acid (NA) or nicotinamide (Nam or NM) is required to prevent pellagra, pharmacological doses of nicotinic acid (NA) can be as high as 2-4 grams. Despite the >100-fold difference in effective dose between pellagra prevention and treatment of dyslipidemias, the beneficial effects of nicotinic acid (NA) on plasma lipids depend on function of nicotinic acid (NA) as an NAD+-boosting compound. See Belenky et al., 2007. According to this view, sirtuin activation would likely be part of the mechanism because nicotinamide (Nam or NM) is an NAD+ precursor in most cells but is a sirtuin inhibitor at high doses. See Kevin J. Bitternnan et al., Inhibition of Silencing and Accelerated Aging by Nicotinamide, a Putative Negative Regulator of Yeast Sir2 and Human SIRT1, 277 J. BIOL. CHEM. 45099 (2002), incorporated by reference herein in its entirety. See also Zamporlini et al., 2014; Mori et al., 2014.
As discussed above, the main NAD+ precursors that feed the Preiss-Handler salvage pathway and other salvage pathways are nicotinamide (Nam or NM) and nicotinamide riboside (NR). See Bogan & Brenner, 2008. Further, studies have shown that nicotinamide riboside (NR) is used in a conserved salvage pathway that leads to NAD+ synthesis through the formation of nicotinamide mononucleotide (NMN). Upon entry into the cell, nicotinamide riboside (NR) is phosphorylated by the NR kinases (“NRKs”), generating nicotinamide mononucleotide (NMN), which is then converted to NAD+ by nicotinic acid/nicotinamide mononucleotide adenylyltransferase (NMNAT). See Bogan & Brenner, 2008. Because nicotinamide mononucleotide (NMN) is the only metabolite that can be converted to NAD+ in mitochondria, nicotinamide (Nam or NM) and nicotinamide riboside (NR) are the two candidate NAD+ precursors that can replenish NAD+ and thus improve mitochondrial fuel oxidation. A key difference is that nicotinamide riboside (NR) has a direct two-step pathway to NAD+ synthesis that bypasses the rate-limiting step of the salvage pathway, nicotinamide phosphoribosyltransferase (NAMPT). Nicotinamide (Nam or NM) requires NAMPT activity to produce NAD+. This reinforces the fact that nicotinamide riboside (NR) is a very effective NAD+ precursor. Conversely, deficiency in dietary NAD+ precursors and/or tryptophan (Trp) causes pellagra. See Bogan & Brenner, 2008. In summary, NAD+ is required for normal mitochondrial function, and because mitochondria are the powerhouses of the cell, NAD+ is required for energy production within cells.
NAD+ was initially characterized as a co-enzyme for oxidoreductases. Though conversions between NAD+, NADH, NADP+, and NADPH would not be accompanied by a loss of total co-enzyme, it was discovered that NAD+ is also turned over in cells for unknown purposes. See Morelly L. Maayan, NAD+-Glycohydrolase of Thyroid Homogenates, 2014 NATURE 1169 (1964), incorporated by reference herein in its entirety. Sirtuin enzymes such as Sir2 of S. cerevisiae and its homologs deacetylate lysine residues with consumption of an equivalent of NAD+, and this activity is required for Sir2 function as a transcriptional silencer. See S. Imai et al., Sir2: An NAD-dependent Histone Deacetylase That Connects Chromatin Silencing, Metabolism, and Aging, 65 COLD SPRING HARBOR SYMPOSIA ON QUANTITATIVE BIOLOGY 297 (2000), incorporated by reference herein in its entirety. NAD+-dependent deacetylation reactions are required, not only for alterations in gene expression, but also for repression of ribosomal DNA recombination and extension of lifespan in response to calorie restriction. See Lin et al., Requirement of NAD and SIR2 for Life-Span Extension by Calorie Restriction in Saccharomyces cerevisiae, 289 SCIENCE 2126 (2000); Lin et al., Calorie restriction extends Saccharomyces cerevisiae lifespan by increasing respiration, 418 NATURE 344 (2002); each of which is incorporated by reference herein in its entirety. NAD+ is consumed by Sir2 to produce a mixture of 2′- and 3′-O-acetylated ADP-ribose plus nicotinamide (Nam or NM) and the deacetylated polypeptide. See Anthony A. Sauve et al., Chemistry of Gene Silencing: the Mechanism of NAD+-Dependent Deacetylation Reactions, 40 BIOCHEMISTRY 15456 (2001), incorporated by reference herein in its entirety. Additional enzymes, including poly(ADP-ribose) polymerases and cADP-ribose synthases are also NAD+-dependent and produce nicotinamide (Nam or NM) and ADP-ribosyl products. See Mathias Ziegler, New functions of a long-known molecule, 267 FEBS J. 1550 (2000); Alexander Birkle, Physiology and pathophysiology of poly(ADP-ribosyl)ation, 23 BIOESSAYS 795 (2001); each of which is incorporated by reference herein in its entirety.
The non-coenzymatic properties of NAD+ have renewed interest in NAD+ biosynthesis. Based on the ability of nicotinamide riboside (NR) to elevate NAD+ synthesis, increase sirtuin activity, and extend lifespan in yeast, nicotinamide riboside (NR) has been employed in mice to elevate NAD+ metabolism and improve health in models of metabolic stress. See Peter Belenky et al., Nicotinamide Riboside Promotes Sir2 Silencing and Extends Lifespan via Nrk and Urh1/Pnp1/Meu1 Pathways to NAD+, 129 CELL 473 (2007), incorporated by reference herein in its entirety. See also Bieganoski & Brenner, 2004. Notably, nicotinamide riboside (NR) allowed mice to resist weight gain on a high-fat diet, and to prevent noise-induced hearing loss. See Carles Cantó et al., The NAD+Precursor Nicotinamide Riboside Enhances Oxidative Metabolism and Protects against High-Fat Diet-Induced Obesity, 15 CELL METABOLISM 838 (2012); Kevin D. Brown et al., Activation of SIRT3 by the NAD+Precursor Nicotinamide Riboside Protects from Noise-Induced Hearing Loss, 20 CELL METABOLISM 1059 (2014); each of which is incorporated by reference herein in its entirety. Data indicate that nicotinamide riboside (NR) is a mitochondrially favored NAD+ precursor and, indeed, in vivo activities of nicotinamide riboside (NR) have been interpreted as depending upon mitochondrial sirtuin activities, though not to the exclusion of nucleocytosolic targets. Andrey Nikiforov et al., Pathways and Subcellular Compartmentation of NAD Biosynthesis in Human Cells, 286 J. BIOLOGICAL CHEM. 21767 (2011); Charles Brenner, Boosting NAD to Spare Hearing, 20 CELL METABOLISM 926 (2014); Carles Cantó et al., NAD+Metabolism and the Control of Energy Homeostasis: A Balancing Act between Mitochondria and the Nucleus, 22 CELL METABOLISM 31 (2015); each of which is incorporated by reference herein in its entirety. Similarly, nicotinamide mononucleotide (NMN), the phosphorylated form of nicotinamide riboside (NR), has been used to treat declining NAD+ in mouse models of overnutrition and aging. See J. Yoshino et al., 2011; A. P. Gomes et al., 2013. Because of the abundance of NAD+-dependent processes, it is not known to what degree NAD+-boosting strategies are mechanistically dependent upon particular molecules such as SIRT1 or SIRT3. In addition, the quantitative effect of nicotinamide riboside (NR) on the NAD+ metabolome has not been reported in any system.
In conclusion, vitamins B1, B2, B3, and B6 are closely intertwined in their biosynthetic pathways, with the maintenance and regeneration of the NADPH intracellular pool depending on the availability of ThDP (vitamin B1), FAD (vitamin B2), and PLP (vitamin B6), along with that of ATP. Critically, the latter is produced through NAD+-dependent OXPHOS and glycolysis, and is necessary for the functionalization of the vitamins B1, B2, and B6 to ThDP, FAD, and PLP, respectively. A shortage of any of these vitamins would impact negatively on the biology of the others. Maximizing these vitamins' bioavailabilities is achieved by conjugating these vitamins to NR, NAR, —NRH, or NARH, or their related derivatives, and by using the NR/NAR uptake to achieve improved vitamin bioavailability.
The compounds and derivatives of the present invention, or salts, hydrates, or solvates thereof, aim at modulating the absorption of vitamins or bioactive compounds of known therapeutic and nutraceutical value by conjugating said vitamins or bioactive compounds to specific B3 vitamins, more specifically NAR, —NR, NARH, —NRH, NMN, NaMN, NMNH, and NaMNH, and partial derivatives thereof.
The compounds and derivatives of the present invention, or salts, hydrates, or solvates thereof, provide improvements on the individual nutrients and B-vitamins in terms of modulating their bioavailabilities.
The compounds and derivatives of the present invention, or salts, hydrates, or solvates thereof, can be used to reduce the risk of developing symptoms, diseases, disorders, or conditions associated with, or having etiologies involving, vitamin B3 deficiencies and/or that would benefit from increased mitochondrial activity, as the key component is nicotinic acid riboside (NAR).
Alcoholism, its link to deficiencies of B-vitamins, and psychological outcomes remain a primary area of research in the modern developed world. Studies have indicated that in patients with alcoholic pellagra, vitamin B3 deficiency may be an important factor influencing both the onset and severity of the condition, and patients with alcoholism are being recommended vitamin B1 as a supplement to minimize dementia and psychological episodes associated with alcohol abuse. Yet combination supplementations are not yet considered, as the pharmacological network between vitamins B1 and B3, in terms of NAD+ bioavailability, has never been demonstrated. This invention aims at tackling vitamin B1/B3 synergistic deficiencies in this class of patients. See W. Todd Penberthy & James B. Kirkland, Niacin, in PRESENT KNOWLEDGE IN NUTRITION 293 (10th ed., 2012), incorporated by reference herein in its entirety.
Mitochondria are critical for the survival and proper function of almost all types of eukaryotic cells. Mitochondria in virtually any cell type can have congential or acquired defects that affect their function. Thus, the clinically significant signs and symptoms of mitochondrial defects affecting respiratory chain function are heterogeneous and variable, depending on the distribution of defective mitochondria among cells and the severity of their deficits, and upon physiological demands upon the affected cells. Nondividing tissues with high energy requirements, e.g., nervous tissue, skeletal muscle, and cardiac muscle are particularly susceptible to mitochondrial respiratory chain dysfunction, but any organ system can be affected.
Symptoms, diseases, disorders, and conditions associated with mitochondrial dysfunction include symptoms, diseases, disorders, and conditions in which deficits in mitochondrial respiratory chain activity contribute to the development of pathophysiology of such symptoms, diseases, disorders, or conditions in a mammal. This includes congenital genetic deficiencies in activity of one or more components of the mitochondrial respiratory chain, wherein such deficiencies are caused by a) elevated intracellular calcium; b) exposure of affected cells to nitric oxide; c) hypoxia or ischemia; d) microtubule-associated deficits in axonal transport of mitochondria; or e) expression of mitochondrial uncoupling proteins.
Symptoms, diseases, disorders, or conditions that would benefit from increased mitochondrial activity generally include for example, diseases in which free radical mediated oxidative injury leads to tissue degeneration, diseases in which cells inappropriately undergo apoptosis, and diseases in which cells fail to undergo apoptosis. Exemplary symptoms, diseases, disorders, or conditions that would benefit from increased mitochondrial activity include, for example, AMDF (Ataxia, Myoclonus and Deafness), auto-immune disease, cancer, CIPO (Chronic Intestinal Pseudoobstruction with myopathy and Ophthalmoplegia), congenital muscular dystrophy, CPEO (Chronic Progressive External Ophthalmoplegia), DEAF (Maternally inherited DEAFness or aminoglycoside-induced DEAFness), DEMCHO (Dementia and Chorea), diabetes mellitus (Type I or Type II), DID-MOAD (Diabetes Insipidus, Diabetes Mellitus, Optic Atrophy, Deafness), DMDF (Diabetes Mellitus and Deafness), dystonia, Exercise Intolerance, ESOC (Epilepsy, Strokes, Optic atrophy, and Cognitive decline), FBSN (Familial Bilateral Striatal Necrosis), FICP (Fatal Infantile Cardiomyopathy Plus, a MELAS-associated cardiomyopathy), GER (Gastrointestinal Reflux), HD (Huntington's Disease), KSS (Kearns Sayre Syndrome), “later-onset” myopathy, LDYT (Leber's hereditary optic neuropathy and DYsTonia), Leigh's Syndrome, LHON (Leber Hereditary Optic Neuropathy), LIMM (Lethal Infantile Mitochondrial Myopathy), MDM (Myopathy and Diabetes Mellitus), MELAS (Mitochondrial Encephalomyopathy, Lactic Acidosis, and Stroke-like episodes), MEPR (Myoclonic Epilepsy and Psychomotor Regression), MERME (MERRF/MELAS overlap disease), MERRF (Myoclonic Epilepsy and Ragged Red Muscle Fibers), MHCM (Maternally Inherited Hypertrophic CardioMyopathy), MICM (Maternally Inherited CardioMyopathy), MILS (Maternally Inherited Leigh Syndrome), Mitochondrial Encephalocardiomyopathy, Mitochondrial Encephalomyopathy, MM (Mitochondrial Myopathy), MMC (Maternal Myopathy and Cardiomyopathy), MNGIE (Myopathy and external ophthalmoplegia, Neuropathy, Gastro-Intestinal, Encephalopathy), Multisystem Mitochondrial Disorder (myopathy, encephalopathy, blindness, hearing loss, peripheral neuropathy), NARP (Neurogenic muscle weakness, Ataxia, and Retinitis Pigmentosa; alternate phenotype at this locus is reported as Leigh Disease), Pearson's Syndrome, PEM (Progressive Encephalopathy), PEO (Progressive External Ophthalmoplegia), PME (Progressive Myoclonus Epilepsy), PMPS (Pearson Marrow-Pancreas Syndrome), psoriasis, RTT (Rett syndrome), schizophrenia, SIDS (Sudden Infant Death Syndrome), SNHL (Sensorineural Hearing Loss), Varied Familial Presentation (clinical manifestations range from spastic paraparesis to multisystem progressive disorder & fatal cardiomyopathy to truncal ataxia, dysarthria, severe hearing loss, mental regression, ptosis, ophthalmoparesis, distal cyclones, and diabetes mellitus), or Wolfram syndrome.
Other symptoms, diseases, disorders, and conditions that would benefit from increased mitochondrial activity include, for example, Friedreich's ataxia and other ataxias, amyotrophic lateral sclerosis (“ALS”) and other motor neuron diseases, macular degeneration, epilepsy, Alpers syndrome, Multiple mitochondrial DNA deletion syndrome, MtDNA depletion syndrome, —Complex I deficiency, Complex II (SDH) deficiency, Complex III deficiency, Cytochrome c oxidase (“COX,” Complex IV) deficiency, Complex V deficiency, Adenine Nucleotide Translocator (“ANT”) deficiency, Pyruvate dehydrogenase (“PDH”) deficiency, Ethylmalonic aciduria with lactic academia, Refractory epilepsy with declines during infection, Autism with declines during infection, Cerebral palsy with declines during infection, materially inherited thrombocytopenia and leukemia syndrome, MARIAHS syndrome (Mitochondrial ataxia, recurrent infections, aphasia, hypouricemia/hypomyelination, seizures, and dicarboxylic aciduria), ND6 dystonia, Cyclic vomiting syndrome with declines during infection, 3-Hydroxy isobutyric aciduria with lactic academia, Diabetes mellitus with lactic acidemia, Uridine responsive neurologic syndrome (“URNS”), Dilated cardiomyopathy, Splenic Lymphoma, or Renal Tubular Acidosis/Diabetes/Ataxis syndrome.
Other symptoms, diseases, disorders, and conditions associated with mitochondrial disorders include, but are not limited to, Post-traumatic head injury and cerebral edema, Stroke, Lewy body dementia, Hepatorenal syndrome, Acute liver failure, NASH (non-alcoholic steatohepatitis), Anti-metastasis/prodifferentiation therapy of cancer, Idiopathic congestive heart failure, Atrial fibrillation (non-valvular), Wolff-Parkinson-White Syndrome, Idiopathic heart block, Prevention of reperfusion injury in acute myocardial infarctions, Familial migraines, Irritable bowel syndrome, Secondary prevention of non-Q wave myocardial infarctions, Premenstrual syndrome, Prevention of renal failure in hepatorenal syndrome, Anti-phospholipid antibody syndrome, Eclampsia/pre-eclampsia, Ischemic heart disease/Angina, and Shy-Drager and unclassified dysautonomia syndromes.
Common symptoms of mitochondrial diseases include cardiomyopathy, muscle weakness and atrophy, developmental delays (involving motor, language, cognitive, or executive function), ataxia, epilepsy, renal tubular acidosis, peripheral neuropathy, optic neuropathy, autonomic neuropathy, neurogenic bowel dysfunction, sensorineural deafness, neurogenic bladder dysfunction, dilating cardiomyopathy, hepatic failure, lactic acidemia, and diabetes mellitus.
Diseases or disorders that would benefit from increased mitochondrial activity include, but are not limited to, neuromuscular disorders (e.g., Friedreich's Ataxia, muscular dystrophy, multiple sclerosis, etc.), disorders of neuronal instability (e.g., seizure disorders, migraine, etc.), developmental delay, ischemia, renal tubular acidosis, chemotherapy fatigue, mitochondrial myopathies, mitochondrial damage (e.g., calcium accumulation, excitotoxicity, nitric oxide exposure, hypoxia, etc.), and mitochondrial deregulation.
A gene defect underlying Friedreich's Ataxia (“FA”), the most common hereditary ataxia, was recently identified and is designated “frataxin.” In FA, after a period of normal development, deficits in coordination develop that progress to paralysis and death, typically between the ages of 30 and 40. The tissues affected most severely are the spinal cord, peripheral nerves, myocardium, and pancreas. Patients typically lose motor control and are confined to wheel chairs, and are commonly afflicted with heart failure and diabetes. The genetic basis for FA involves GAA trinucleotide repeats in an intron region of the gene encoding frataxin. The presence of these repeats results in reduced transcription and expression of the gene. Frataxin is involved in regulation of mitochondrial iron content. When cellular frataxin content is subnormal, excess iron accumulates in mitochondria, promoting oxidative damage and consequent mitochondrial degeneration and dysfunction. When intermediate numbers of GAA repeats are present in the frataxin gene intron, the severe clinical phenotype of ataxia may not develop. However, these intermediate-length trinucleotide extensions are found in 25% to 30% of patients with non-insulin dependent diabetes mellitus, compared to about 5% of the nondiabetic population.
Muscular dystrophy refers to a family of diseases involving deterioration of neuromuscular structure and function, often resulting in atrophy of skeletal muscle and myocardial dysfunction. In the case of Duchenne muscular dystrophy, mutations or deficits in a specific protein, dystrophin, are implicated in its etiology. Mice with their dystrophin genes inactivated display some characteristics of muscular dystrophy, and have an approximately 50% deficit in mitochondrial respiratory chain activity. A final common pathway for neuromuscular degeneration, in most cases, is calcium-mediated impairment of mitochondrial function.
Epilepsy is often present in patients with mitochondrial cytopathies, involving a range of seizure severity and frequency, e.g., absence, tonic, atonic, myoclonic, and status epilepticus, occurring in isolated episodes or many times daily.
Delays in neurological or neuropsychological development are often found in children with mitochondrial diseases. Development and remodeling of neural connections requires intensive biosynthetic activity, particularly involving synthesis of neuronal membranes and myelin, both of which require pyrimidine nucleotides as cofactors. Uridine nucleotides are involved in activation and transfer of sugars to glycolipids and glycoproteins. Cytidine nucleotides are derived from uridine nucleotides, and are crucial for synthesis of major membrane phospholipid constituents like phosphatidylcholine, which receives its choline moiety from cytidine diphosphocholine. In the case of mitochondrial dysfunction (due to either mitochondrial DNA defects or any of the acquired or conditional deficits like excitotoxic or nitric oxide-mediated mitochondrial dysfunction) or other conditions resulting in impaired pyrimidine synthesis, cell proliferation and axonal extension are impaired at crucial stages in development of neuronal interconnections and circuits, resulting in delayed or arrested development of neuropsychological functions like language, motor, social, executive function, and cognitive skills. In autism, for example, magnetic resonance spectroscopy measurements of cerebral phosphate compounds indicate that there is global undersynthesis of membranes and membrane precursors indicated by reduced levels of uridine diphosphosugars, and cytidine nucleotide derivatives involved in membrane synthesis. Disorders characterized by developmental delay include Rett's Syndrome, pervasive developmental delay (or PDD-NOS “pervasive developmental delay not otherwise specified” to distinguish it from specific subcategories like autism), autism, Asperger's Syndrome, and Attention Deficit/Hyperactivity Disorder (“ADHD”), which is becoming recognized as a delay or lag in development of neural circuitry underlying executive functions.
Oxygen deficiency results in both direct inhibition of mitochondrial respiratory chain activity by depriving cells of a terminal electron acceptor for Cytochrome c reoxidation at Complex IV, and indirectly, especially in the nervous system, via secondary post-anoxic excitotoxicity and nitric oxide formation. In conditions like cerebral anoxia, angina or sickle cell anemia crises, tissues are relatively hypoxic. In such cases, compounds that increase mitochondrial activity provide protection of affected tissues from deleterious effects of hypoxia, attenuate secondary delayed cell death, and accelerate recovery from hypoxic tissue stress and injury.
Acidosis due to renal dysfunction is often observed in patients with mitochondrial disease, whether the underlying respiratory chain dysfunction is congenital or induced by ischemia or cytotoxic agents like cisplatin. Renal tubular acidosis often requires administration of exogenous sodium bicarbonate to maintain blood and tissue pH.
Mitochondrial DNA damage is more extensive and persists longer than nuclear DNA damage in cells subjected to oxidative stress or cancer chemotherapy agents like cisplatin due to both greater vulnerability and less efficient repair of mitochondrial DNA. Although mitochondrial DNA may be more sensitive to damage than nuclear DNA, it is relatively resistant, in some situations, to mutagenesis by chemical carcinogens. This is because mitochondria respond to some types of mitochondrial DNA damage by destroying their defective genomes rather than attempting to repair them. This results in global mitochondrial dysfunction for a period after cytotoxic chemotherapy. Clinical use of chemotherapy agents like cisplatin, mitomycin, and cytoxan is often accompanied by debilitating “chemotherapy fatigue,” prolonged periods of weakness and exercise intolerance that may persist even after recovery from hematologic and gastrointestinal toxicities of such agents.
Mitochondrial myopathies range from mild, slowly progressive weakness of the extraocular muscles to severe, fatal infantile myopathies and multisystem encephalomyopathies. Some syndromes have been defined, with some overlap between them. Established syndromes affecting muscle include progressive external ophthalmoplegia, the Kearns-Sayre syndrome (with ophthalmoplegia, pigmentary retinopathy, cardiac conduction defects, cerebellar ataxia, and sensorineural deafness), the MELAS syndrome (mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes), the MERFF syndrome (myoclonic epilepsy and ragged red fibers), limb-girdle distribution weakness, and infantile myopathy (benign or severe and fatal). Muscle biopsy specimens stained with modified Gomori's trichrome stain show ragged red fibers due to excessive accumulation of mitochondria. Biochemical defects in substrate transport and utilization, the Krebs cycle, oxidative phosphorylation, or the respiratory chain are detectable. Numerous mitochondrial DNA point mutations and deletions have been described, transmitted in a maternial, nonmendlian inheritance pattern. Mutations in nuclear-encoded mitochondrial enzymes occur.
A fundamental mechanism of cell injury, especially in excitable tissues, involves excessive calcium entry into cells, as a result of either leakage through the plasma membrane or defects in intracellular calcium handling mechanisms. Mitochondria are major sites of calcium sequestration, and preferentially utilize energy from the respiratory chain for taking up calcium rather than for ATP synthesis, which results in a downward spiral of mitochondrial failure, because calcium uptake into mitochondria results in diminished capabilities for energy transduction.
Excessive stimulation of neurons with excitatory amino acids is a common mechanism of cell death or injury in the central nervous system. Activation of glutamate receptors, especially of the subtype designated NMDA receptors, results in mitochondrial dysfunction, in part through elevation of intracellular calcium during excitotoxic stimulation. Conversely, deficits in mitochondrial respiration and oxidative phosphorylation sensitizes cells to excitotoxic stimuli, resulting in cell death or injury during exposure to levels of excitotoxic neurotransmitters or toxins that would be innocuous to normal cells.
Nitric oxide (about 1 micromolar) inhibits cytochrome oxidase (Complex IV) and thereby inhibits mitochondrial respiration; moreover, prolonged exposure to nitric oxide (NO) irreversibly reduces Complex I activity. Physiological or pathophysiological concentrations of NO thereby inhibit pyrimidine biosynthesis. Nitric oxide is implicated in a variety of neurodegenerative disorders including inflammatory and autoimmune diseases of the central nervous system, and is involved in mediation of excitotoxic and post-hypoxic damage to neurons.
Oxygen is the terminal electron acceptor in the respiratory chain. Oxygen deficiency impairs electron transport chain activity, resulting in diminished pyrimidine synthesis as well as diminished ATP synthesis via oxidative phosphorylation. Human cells proliferate and retain viability under virtually anaerobic conditions if provided with uridine and pyruvate (or a similarly effective agent for oxidizing NADH to optimize glycolytic ATP production).
Transcription of mitochondrial DNA encoding respiratory chain components requires nuclear factors. In neuronal axons, mitochondria must shuttle back and forth to the nucleus in order to maintain respiratory chain activity. If axonal transport is impaired by hypoxia or by drugs like taxol that affect microtubule stability, mitochondria distant from the nucleus undergo loss of cytochrome oxidase activity.
Mitochondria are the primary source of free radicals and reactive oxygen species, due to spillover from the mitochondrial respiratory chain, especially when defects in one or more respiratory chain components impairs orderly transfer of electrons from metabolic intermediates to molecular oxygen. To reduce oxidative damage, cells can compensate by expressing mitochondrial uncoupling proteins (“UCP”), of which several have been identified. UCP-2 is transcribed in response to oxidative damage, inflammatory cytokines, or excess lipid loads, e.g., fatty liver and steatohepatitis. UCPs reduce spillover of reactive oxidative species from mitochondria by discharging proton gradients across the mitochondrial inner membrane, in effect wasting energy produced by metabolism and rendering cells vulnerable to energy stress as a trade-off for reduced oxidative injury.
A rationale for synergy between vitamins B1, B2, B3, and B6 is explained herein. Pairing vitamins B1, B2, or B6 with nicotinamide riboside (NR) is hypothesized to act synergistically on the NAD+ biosynthetic pathway and have a positive effect. This is due to the fact that vitamins B1, B2, and B6 are required for NAD+ biosynthesis through NAMPT-dependent pathways, allowing for the further recycling of nicotinamide (Nam or NM) generated from the NR-produced NAD+. Of all the B3-vitamins, only NR functions independently of NAMPT for NAD+ synthesis, in a mole to mole perspective. See Penberthy & Kirkland, 2012. See also Yuling Chi & Anthony A. Sauve, Nicotinamide riboside, a trace nutrient in foods, is a vitamin B3 with effects on energy metabolism and neuroprotection, 16 CURR. OPINION IN CLIN. NUTRITION & METABOLIC CARE 657 (2013), incorporated by reference herein in its entirety. Additionally, vitamin B2 (FAD precursor) is a key vitamin for mitochondrial fatty acid oxidation and OXPHOS processes. Mitochondrial dysfunction can arise from FAD/FADH2 imbalance or deficiency, and it is hypothesized that pairing vitamin B2 to vitamin B3 NAD-precursors would address multiple pathways of mitochondrial dysfunction.
One embodiment of the compounds and derivatives of this invention, or salts, hydrates, or solvates thereof, is represented by the co-supplementation of NR (or NAR and other vitamin B3 derivatives) with choline. The normal process for eliminating nicotinamide (Nam or NM) from the body is by methylation. It has been known for a long time that mice given high-dose nicotinamide (Nam or NM) display growth inhibition due to a choline deficiency. Co-supplementation of nicotinamide (Nam or NM) and methionine (choline precursor) completely reversed the growth inhibition. See M. Knip et al., Safety of high-dose nicotinamide: a review, 43 DIABETOLOGIA 1337 (2000), incorporated by reference herein in its entirety. One possible underlying mechanism proposes that, in the brain, the methyl group being added to the nicotinamide (Nam or NM) to eliminate it comes from choline (a methyl donor). All of the NAD+ precursors disclosed herein eventually become nicotinamide (Nam or NM), and are then eliminated through this pathway.
Resveratrol and other related sirtuin activators (like pterostilbene, for example) represent another embodiment of the compounds and derivatives of the present invention, or salts, hydrates, or solvates thereof. Sirtuins are NAD+-dependent enzymes that play vital roles in protecting the genome through histone deacetylation. Several reports have shown that sirtuins play a role in lifespan/healthspan of an organism, and the activity of sirtuins requires available NAD+. See Penberthy & Kirkland, 2012. Thus, resveratrol (and similar compounds), which can induce sirtuin expression, requires a concomitant increase in NAD+ availability in order to realize the increased sirtuin activity. Preferably, resveratrol (and/or similar compounds) is co-supplemented with one of the NAD+-precursors reported herein.
One embodiment of the compounds and derivatives of the present invention, or salts, hydrates, or solvates thereof, is represented by the products formed as a result of joining the nicotinic acid (NA) ester at the 5′-hydroxy of NR and NR, and the corresponding reduced forms thereof. Synergistic effects of nicotinate and NR (or derivatives thereof) are anticipated. Nicotinic acid (NA) and nicotinamide riboside (NR) use different pathways to both ultimately induce NAD+ levels.
Another embodiment of the compounds and derivatives of the present invention, or salts, hydrates, or solvates thereof, is represented by the derivatives of all of these nicotinoyl riboside conjugates and reduced nicotinoyl riboside conjugates, or salts, hydrates, or solvates thereof.
It is expected that certain conjugate molecules will have better physiochemical properties than the parent molecules. This increased stability will be observed in formulations and during the digestive process or other breakdown processes for these molecules (in the blood/cells after oral or topical delivery). The improved properties are advantageous for certain formulation and delivery applications. For instance, it is considered that non-specific plasma protein binding of such derivatives will decrease and allow for increased concentration of free circulating NAD+ precursors.
If new compounds and derivatives comprising nicotinoyl riboside conjugates and reduced nicotinoyl riboside conjugates could be found, this would represent a useful contribution to the art. Furthermore, if new methods of preparing compounds and derivatives comprising nicotinoyl riboside conjugates and reduced nicotinoyl riboside conjugates, or salts, hydrates, or solvates thereof, could be found, this would also represent a useful contribution to the art.